A method for the isolation and purification of normal rat liver and hepatoma nuclear “ghosts” by zonal centrifugation

Abstract

A method is presented for the isolation of nuclear membranes in the form of large fragments and intact nuclear “ghosts” from normal rat liver and an aminoazo dye induced rat hepatoma. The preparation involves overnight incubation at 4°C of the nuclear pellet suspension from hypotonic homogenates, followed by rate-dependent fractionation on a sucrose gradient in an A-XII Zonal rotor. The fraction sedimenting between mitochondria and nuclei was observed using phase contrast microscopy to be rich in large membrane fragments and nuclear “ghosts”. Isopycnic centrifugation of this fraction on sucrose gradients, in conventional swinging-bucket and zonal rotors resulted in a purification of the nuclear membranes. No significant difference in the density of normal rat liver and hepatoma nuclear membranes was detected. The purity and homogeneity of the nuclear membranes was confirmed by electron microscopy using both thin sectioning and negative contrast staining. Both electron microscopic techniques revealed the presence of pore complexes on the nuclear membranes, there being a marked absence of membranes not displaying these morphological features in the purified preparation. An assessment of the degree of contamination of the membranes with chromatin was obtained by thin sectioning and by measurement of DNA: protein ratios in purified preparations of both nuclear membranes and intact nuclei. The method developed for the preparation of nuclear membranes is an extension of those devised for the isolation of plasma membranes under hypotonic conditions, and the preparative procedures are discussed in relation to previous studies upon both plasma membranes and nuclear membranes. The method is thought to be significant as an addition to the use of rate and isopycnic zonal centrifugation for the purification of cellular organelles.